TMEM16F scramblase regulates angiogenesis via endothelial intracellular signaling

Abstract

TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.

Keywords: Angiogenesis; Endothelial cells; Scramblase; Src; TMEM16F; VE-cadherin.

Fig. 1.

TMEM16F is functionally expressed in HUVECs. (A) qRT-PCR of TMEM16F in control and TMEM16F knockdown HUVECs (n=7, from five biological replicates). mRNA expression is normalized to GAPDH expression. (B) Representative western blots of TMEM16F in HUVECs with control or TMEM16F siRNA knockdown. (C) Densitometry quantifications of TMEM16F and loading control β-actin (n=4, from four biological replicates). (D) Schematic of the fluorescence-based scrambling assay. PS, phosphatidylserine; AnV, Annexin V. (E) Representative images of Ca²⁺ and AnV in control (left) and TMEM16F knockdown (right) HUVECs stimulated with 2.5 μM ionomycin. (F,G) Quantifications of the time course (F) or the maximum fluorescence intensity of AnV at 10 min post ionomycin stimulation (G) for control siRNA (n=37) and TMEM16F (n=46) (from four biological replicates). Each dot represents AnV signals from one cell. (H) Representative currents recorded in control or TMEM16F siRNA knockdown HUVECs. The currents were elicited by a voltage step protocol from −100 mV to +160 mV with a 20 mV increment. Holding potential was set at −60 mV. (I) Current-voltage (I-V) relationship of currents recorded in H (n=13 for control siRNA and n=14 for TMEM16F siRNA, from two biological replicates). Data are presented as mean±s.e.m. ****P<0.0001 (unpaired two-tailed t-test). a.u., arbitrary units.

Fig. 2.

TMEM16F deficiency suppresses angiogenesis in vivo. (A,B) IB4 (green) staining of retinal whole-mounts from P5 wild-type (WT, A) and Tmem16F knockout (KO, B) littermates. White boxes indicate enlarged area shown in the middle panels. The bottom panels illustrate processed enlarged area used for the quantification of loop numbers and hole areas. White areas are defined as holes and vasculature (black lines) encircling the holes as loops. (C–E) Quantification of retinal vasculature coverage (C), average hole areas (D) and number of loops (E) calculated from four random fields of each retina. Data are presented as mean±s.e.m. Each dot represents data from one animal (n=7 pairs of WT and Tmem16F KO littermates). *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed t-test).

Fig. 3.

TMEM16F deficiency in HUVEC impairs tube formation in vitro. (A,B) Representative images of tube formation for control and TMEM16F knockdown HUVECs (A), and TMEM16F knockdown HUVECs overexpressing wild-type (WT) and D703R loss-of-function mTmem16F (B). Yellow arrowheads indicate thin and stretched tubes. (C) Quantification of loop numbers at 24 h after seeding (control and TMEM16F knockdown, n=20 each; WT and D703R mTmem16F overexpressed in TMEM16F knockdown HUVECs, n=19 each; from three biological replicates). Each dot represents data from one well of tube formation μ-Slide. Data are presented as mean±s.e.m. n.s.; non-significant; **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA with Tukey's multiple comparisons test).

Fig. 4.

TMEM16F deficiency in HUVECs disrupts intracellular Src-VE-cadherin signaling. (A) qRT-PCR of VE-cadherin in control and TMEM16F knockdown HUVECs (n=7 from five biological replicates). The gene expression is normalized to GAPDH expression. **P<0.01 (unpaired two-tailed t-test). (B,C) Representative western blot of anti-VE-cadherin (B) and densitometry quantifications (n=9 from none biological replicates) (C). (D,E) Representative western blot of anti-Tyr685 phosphorylated and anti-total VE-cadherin (D) and densitometry quantifications (n=5 from five biological replicates) (E). (F,G) Representative western blot anti-Tyr416 phosphorylated and anti-total Src (F) and densitometry quantifications (n=4 from four biological replicates) (G). (H,I) TMEM16F knockdown increases Src phosphorylation without or with 100 ng/ml of VEGF stimulation. (H) Proximity ligation assay with antibodies against Src and p-SFK(Tyr416) (pSrc/Src, red) in HUVECs without (top) or with (bottom) 100 ng/ml of VEGF stimulation for 5 min. Cell junctions were stained for VE-cadherin (green) and nuclei with DAPI (blue). Enlarged views of the dotted boxes are shown on the right. (I) Mean fluorescence intensity quantifications of junctional PLA signals (n=6 from four biological replicates). (J) FITC–dextran (40 kDa, 1 mg/ml) Transwell assay (n=9 from three biological replicates). **P<0.01, ***P<0.001, ****P<0.0001 [unpaired two-tailed t-test (A,C,E,G); two-way ANOVA with Tukey's multiple comparisons test (I,J)]. All data are presented as mean±s.e.m. a.u., arbitrary units. (K) Cartoon illustration of the intracellular signaling role of TMEM16F in angiogenesis. TMEM16F-mediated PS exposure limits Src membrane association and phosphorylation, maintaining VE-cadherin membrane expression and normal angiogenesis (top). TMEM16F deficiency increases Src membrane association and phosphorylation and promotes its activation, which downregulates VE-cadherin and leads to defective angiogenesis (bottom).